2.4. Experimental design
Experiments were performed in 100-ml Erlenmeyer flasks containing
20 ml of liquid medium plus 1mM chrysene dissolved in
DMF to 1 ml. In addition, as the strains have different growth rates,
the period of incubation was varied from 5 to 7 days in order to
obtain a similar radial growth and to minimize variation in the starting
inoculums.Mycelia plugs of a selected funguswere cut fromthe
outer edge of an actively growing culture on an inoculums plate.
Three5-mmdisks obtained by punching out with a cork-borer from
the outer edge of an actively growing culture of a particular fungus
were inoculated into a flask containing 20 ml of liquid medium
supplemented with 1mM of substrates. The flasks were incubated
at 25 ◦C. Growth and substrate consumptionwere determined at 7-
day intervals. One set of inoculated flaskswas incubated stationary.
The effect of different carbon source concentrations on the degradation
of chrysene was studied using glucose in the concentration
range 2–10%. The effect of varying the nitrogen source concentration
on the degradation of chrysene was studied by replacing
ammonium tartrate with polypeptone in the concentration range
2–10%. The effect of varying the surfactant on chrysene’s degradation
was studied using tween 80 and tween 20. Agitation at
120rpm was conducted to enhance the degradation of chrysene
in the liquid medium. All media were sterilized by autoclaving at
121 ◦C for 20 min. Control experiments were performed by incubating
chrysene in autoclaved cultures (121 ◦C for 20 min) and by
incubating MSB medium with chrysene without an inoculum. All
assayswere conducted in triplicate. Before the incubation, a flask of
each treatment was selected for immediate extraction. All remaining
flaskswere incubated for 15 and 30 days. The culture brothwas
blendedwith ethyl acetate to extract the aromatic hydrocarbon and
metabolites from the mycelia.